The use of both vaccination and antibiotic therapy is the most effective approach against inhalational anthrax. Unfortunately, there are two significant problems associated with current post-exposure prophylaxis (PEP): 1) the limitations of the anthrax vaccine adsorbed (AVA) which requires multiple needle injections by health professionals to achieve a protective immunity and 2) the concern over adverse effects of the long-term antibiotic treatment. It is essential to develop a simple and effective approach against post-exposure anthrax. Vaxin is developing a replication competent adenovirus-free, non-replicating adenovirus 5 (Ad5)-vectored nasal anthrax vaccine encoding a humanized wild type (wt) B. anthraxcis protective antigen (PA) gene (AdwtPA) and has demonstrated that: 1) protection with single dose; 2) excellent safety profile in animals; 3) easy, patient- friendly, needle-free administration; 4) rapid and long-lasting immunity; and 5) its protection potency is not impaired by pre-existing anti-Ad5 immunity. However, AdwtPA expressing wt PA protein may not safe for individual who has been freshly exposed to anthrax spores. Fortunately, numerous studies have demonstrated that dominant negative inhibitory (DNI) PA mutants are not only more immunogenic but also more effective than wt PA as a therapeutic agent. Therefore, it would be reasonable to expect that an Ad5 vector encoding a humanized DNI PA mutant gene (AdDNIPA) could be a better and safer PEP vaccine against anthrax infection. More importantly, use of AdDNIPA as a component of PEP should be able to minimize the time and dose required for post-exposure antibiotic therapy. In this proposal, we will generate a RCA-free AdDNIPA vector expressing secretory PA83 protein with K397D and D425K double mutations as a new nasal anthrax vaccine candidate and evaluate its immunogenicity and the protection efficacy as a PEP vaccine in conjunction with a short-course ciprofloxacin prophylaxis against respiratory anthrax in A/J mice. PUBLIC HEALTH RELEVANCE: Novel approaches for the prophylaxis and treatment of post-exposure anthrax are still required for counteracting the threat posed by bioterrorist attacks. Evidence is emerging that nasal spray of a non-replicating adenovirus serotype 5-derived vectors encoding Bacillus anthracis protective antigen can bypass pre-existing anti-Ad5 immunity and confer immediate protection like a therapeutic agent and elicit rapid and sustained protective immunity as a vaccine. Use of this adenovirus vectored anthrax nasal vaccine as a component of post-exposure prophylaxis will significantly reduce the time and dose required for antibiotic therapy.